Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain fr om the natural restriction enzyme Fokl, Because of the flexibility of DNA r ecognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by similar to 4 bp with a 5' overhang, as for native Fokl, Furthe rmore, amino acid changes in the dimer interface of the cleavage domain abo lished activity. These results reflect a requirement for dimerization of th e cleavage domain. The dependence of cleavage efficiency on the distance be tween two inverted binding sites was determined and both upper and lower li mits were defined, Two different zinc finger combinations binding to non-id entical sites also supported specific cleavage, Molecular modeling was empl oyed to gain insight into the precise location of the cut sites. These resu lts define requirements for effective targets of chimeric nucleases and wil l guide the design of novel specificities for directed DNA cleavage in vitr o and in vivo.